The t(8;21) is one of the most common cytogenetic abnormalities associated with acute myeloid leukemia (AML), present in 12% of de novo AML cases. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (AML1) and ETO (MTG8/RUNX1T1) proteins. RUNX1 is a DNA- binding transcription factor that is essential for regulating hematopoiesis. ETO facilitates numerous protein- protein interactions, notably with NCoR/SIN3A/HDAC transcriptional repression complexes. The RUNX1-ETO fusion protein is therefore capable of promoting leukemogenesis through aberrant transcriptional repression of normal RUNX1 target genes. The identification and characterization of specific genes whose expression is disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21) AML and lead to improved treatment for patients. One such gene that we have identified as being ~30-fold downregulated in a murine model of t(8;21) leukemia is Ras association domain family member 2 (RASSF2). Additionally, we have discovered that RASSF2 expression is consistently reduced in primary human t(8;21) leukemia cells. RASSF2 is a tumor suppressor that is frequently silenced via aberrant promoter hypermethylation in primary solid tumors, but not in adjacent normal tissue. Normal RASSF2 expression levels are highest in myeloid cells, but it has not yet been characterized in the hematopoietic system or in leukemia. Furthermore, our preliminary data demonstrates that RASSF2 functions as a tumor suppressor in leukemia cell lines and in primary bone marrow retrovirally transduced to express RUNX1-ETO. The overall goal of this proposal is to test the hypothesis that RUNX1-ETO binds the RASSF2 promoter to aberrantly repress its transcription and that loss of RASSF2 in t(8;21) hematopoietic cells directly contributes to AML development through disruption of one or more of its downstream signaling mechanisms. Multiple approaches will be taken to identify the mechanism of transcriptional regulation through which RUNX1-ETO is sufficient to repress RASSF2 expression, and whether this repression involves direct binding and recruitment of chromatin remodeling complexes to the promoter. This proposal will then seek to determine the molecular mechanism(s) through which RASSF2 functions as a tumor suppressor in hematopoietic cells in order to assess the biological relevance of losing RASSF2 in t(8;21) AML. This will include the novel characterization of known RASSF2 interaction partners, MST1/2 and PAR-4, in myeloid cells and AML. If loss of RASSF2 is indeed a key step in t(8;21) AML development this study should provide novel insight towards developing targeted therapies involving RASSF2-dependent signaling pathways.